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Abstract:

The present disclosure provides methods and compositions for preventing
or treating MV-induced or disuse-induced skeletal muscle infirmities in a
mammalian subject. The methods further include administering to the
subject an effective amount of an aromatic-cationic peptide.

Claims:

1. A method of treating or preventing skeletal muscle infirmities in a
mammalian subject, comprising administering to the mammalian subject a
therapeutically effective amount of the peptide
D-Arg-2',6'Dmt-Lys-Phe-NH2 or a pharmaceutically acceptable salt
thereof.

7. A method of treating or preventing MV-induced diaphragm dysfunction in
a mammalian subject, comprising administering to the mammalian subject a
therapeutically effective amount of the peptide
D-Arg-2',6'Dmt-Lys-Phe-NH2 or a pharmaceutically acceptable salt
thereof.

8. The method of claim 7, wherein the peptide is administered to the
subject prior to MV, during MV, or both.

15. A method for treating a disease or condition characterized by
increased oxidative damage in skeletal muscle of a mammalian subject in
need thereof, the method comprising: administering to the subject an
effective amount of D-Arg-2',6'Dmt-Lys-Phe-NH2 or a pharmaceutically
acceptable salt thereof, wherein the oxidative damage is associated with
a variation in the gene expression or protein levels, activity, or
degradation of one or more biomarkers selected from the group consisting
of calpain, caspase-3, caspase 12, 20S proteasome, E3 ligases,
atrogin-1/MAFbx, MuRF-1, αII-spectrin, sarcomeric protein,
4-HNE-conjugated cytosolic proteins, and protein carbonyls in
myofibrillar proteins, compared to a control level.

17. The method of claim 15, wherein the control level is the levels of
the one or more biomarkers from a healthy individual not afflicted with
disuse-induced skeletal muscle atrophy or MV-induced diaphragm
dysfunction.

18. The method of claim 15, wherein the peptide is administered to the
subject prior to or during the increased oxidative damage.

20. The method of claim 15, wherein the skeletal muscle comprises soleus
muscle or plantaris muscle, or both soleus and plantaris muscle.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application
No. 61/308,508, filed Feb. 26, 2010, which is incorporated herein by
reference in its entirety.

TECHNICAL FIELD

[0003] Disclosed herein are methods and compositions that include
aromatic-cationic peptides useful for the prevention and treatment of
skeletal muscle infirmities, such as weakness, dysfunction and/or muscle
atrophy. In particular, methods and compositions for the prevention and
treatment of mechanical ventilation (MV)-induced diaphragm infirmities,
and disuse-induced skeletal muscle infirmities are disclosed.

BACKGROUND

[0004] The following description is provided to assist the understanding
of the reader. None of the information provided or references cited is
admitted to be prior art to the present invention.

[0005] Mechanical ventilation (MV) is clinically employed to achieve
adequate pulmonary gas exchange in subjects incapable of maintaining
sufficient alveolar ventilation. Common indications for MV include
respiratory failure, heart failure, surgery, drug overdose, and spinal
cord injuries. Even though MV is a life-saving measure for subjects with
respiratory failure, complications associated with weaning patients from
MV are common. Indeed, weaning difficulties are an important clinical
problem; 20-30% of mechanically ventilated subjects experience weaning
difficulties. The "failure to wean" may be due to several factors
including respiratory muscle weakness of the diaphragm, a skeletal
muscle.

[0006] Skeletal muscle weakness emanate from muscle fiber atrophy and
dysfunction. In this regard, muscle disuse presents a widespread problem
for individuals subject to body or limb immobilization, e.g., muscle
constraints due to bone fracture casting or prolonged MV. Such muscle
disuse, however, does not elucidate the etiology of muscle fiber
degradation at the cellular level. To this end, oxidative stress, such as
the generation of reactive oxygen species (ROS) via xanthine oxidase
activation, may impart a mechanism for skeletal muscle degradation and
contractile dysfunction. However, inhibition of xanthine oxidase activity
does not completely protect against the effects of skeletal muscle
disuse-induced or MV-induced oxidative stress, concomitant atrophy and
weakness. Accordingly, identifying additional factors associated with
muscle dysfunction and atrophy are considerations in the development of
new strategies for preventing or treating these ailments.

SUMMARY

[0007] Disclosed herein are methods and compositions for the prevention
and treatment of skeletal muscle infirmities, such as mechanical
ventilation (MV)-induced diaphragm weakness, dysfunction and/or atrophy.
Generally, the methods and compositions include one or more
aromatic-cationic peptides or a pharmaceutically acceptable salt there
of, (e.g., acetate or trifluoroacetate salt), and in some embodiments, a
therapeutically effective amount of one or more aromatic-cationic
peptides or a pharmaceutically acceptable salt thereof, (e.g., acetate or
trifluoroacetate salt) is administered to a subject in need thereof, to
treat or prevent or treat skeletal muscle infirmity such as weakness,
dysfunction and/or atrophy.

[0008] Disclosed herein are methods and compositions for the prevention
and treatment of skeletal muscle infirmities, such as mechanical
ventilation (MV)-induced diaphragm weakness, dysfunction and/or atrophy,
and/or disuse induced muscle infirmities. Generally, the methods and
compositions include one or more aromatic-cationic peptides or a
pharmaceutically acceptable salt thereof, (e.g., acetate or
trifluoroacetate salt), and in some embodiments, a therapeutically
effective amount of one or more aromatic-cationic peptides or a
pharmaceutically acceptable salt there of, (e.g., acetate or
trifluoroacetate salt) is administered to a subject in need thereof, to
treat or prevent skeletal muscle infirmities.

[0009] In some aspects, methods for treating or preventing skeletal muscle
infirmities in a mammalian subject are provided. Typically, the methods
include administering to the mammalian subject a therapeutically
effective amount of the peptide D-Arg-2',6'Dmt-Lys-Phe-NH2, or a
pharmaceutically acceptable salt thereof, (e.g., acetate or
trifluoroacetate salt). In some embodiments, the peptide is administered
orally, topically, systemically, intravenously, subcutaneously,
intraperitoneally, or intramuscularly.

[0010] In some embodiments, the skeletal muscle comprises diaphragmatic
muscle, and the skeletal muscle infirmity results from mechanical
ventilation (MV). In some embodiments, a method of treating or preventing
MV-induced diaphragm dysfunction in a mammalian subject is provided. In
some embodiments, the duration of the MV is at least 10 hours, and in
some embodiments, the peptide is administered to the subject prior to MV,
during the MV, or both prior to and during the MV. In some embodiments,
the peptide is administered orally, topically, systemically,
intravenously, subcutaneously, intraperitoneally, or intramuscularly

[0011] Additionally or alternatively, in some embodiments, methods of
treating or preventing disuse-induced skeletal muscle atrophy in a
mammalian subject are provided. Typically, such methods include
administering to the mammalian subject a therapeutically effective amount
of the peptide D-Arg-2',6'Dmt-Lys-Phe-NH2 or a pharmaceutically
acceptable salt thereof (e.g., acetate or trifluoroacetate salt). In some
embodiments, the skeletal muscle includes soleus muscle or plantaris
muscle, or both the soleus and plantaris muscle. In some embodiments, the
peptide is administered to the subject prior to or during the disuse. In
some embodiments, the peptide is administered orally, topically,
systemically, intravenously, subcutaneously, intraperitoneally, or
intramuscularly

[0012] Additionally or alternatively, in some embodiments, methods for
treating a disease or condition characterized by increased oxidative
damage in skeletal muscle of a mammalian subject are provided. Typically,
such methods include administering to the subject an effective amount of
D-Arg-2',6'Dmt-Lys-Phe-NH2 or a pharmaceutically acceptable salt
thereof (e.g., acetate or trifluoroacetate salt). In some embodiments,
the peptide is administered to the subject prior to or during the
increased oxidative damage. In some embodiments, the oxidative damage is
associated with a variation in the gene expression or protein levels,
activity, or degradation of one or more biomarkers compared to a control
level. In some embodiments, the control level is the levels of the one or
more biomarkers from a healthy individual not afflicted with
disuse-induced skeletal muscle atrophy or MV-induced diaphragm
dysfunction. In some embodiments, the biomarkers are selected from the
group consisting of calpain, caspase-3, caspase-12, 20S proteasome, E3
ligases, atrogin-1/MAFbx, MuRF-1, αII-spectrin, sarcomeric protein,
4-HNE-conjugated cytosolic proteins, and protein carbonyls in
myofibrillar proteins. In some embodiments, the disease or condition
characterized by increased oxidative damage includes disuse-induced
skeletal muscle atrophy or MV-induced diaphragm dysfunction. In some
embodiments, the peptide is administered orally, topically, systemically,
intravenously, subcutaneously, intraperitoneally, or intramuscularly

[0013] In one aspect, the disclosure provides a method of treating or
preventing MV-induced diaphragm dysfunction, comprising administering to
a mammalian subject in need thereof a therapeutically effective amount of
an aromatic-cationic peptide. In some embodiments, the aromatic-cationic
peptide is a peptide including:

[0014] at least one net positive charge;

[0015] a minimum of four amino acids;

[0016] a maximum of about twenty amino acids;

[0017] a relationship between the minimum number of net positive charges
(pm) and the total number of amino acid residues (r) wherein 3pm, is
the largest number that is less than or equal to r+1; and a relationship
between the minimum number of aromatic groups (a) and the total number of
net positive charges (pt) wherein 2a is the largest number that is less
than or equal to pt+1, except that when a is 1, pt may also be 1. In some
embodiments, the mammalian subject is a human.

[0018] In one embodiment, 2pm, is the largest number that is less
than or equal to r+1, and a may be equal to pt. The aromatic-cationic
peptide may be a water-soluble peptide having a minimum of two or a
minimum of three positive charges.

[0019] In one embodiment, the peptide comprises one or more non-naturally
occurring amino acids, for example, one or more D-amino acids. In some
embodiments, the C-terminal carboxyl group of the amino acid at the
C-terminus is amidated. In certain embodiments, the peptide has a minimum
of four amino acids. The peptide may have a maximum of about 6, a maximum
of about 9, or a maximum of about 12 amino acids.

[0020] In one embodiment, the peptide comprises a tyrosine or a
2',6'-dimethyltyrosine (Dmt) residue at the N-terminus. For example, the
peptide may have the formula Tyr-D-Arg-Phe-Lys-NH2 (SS-01) or
2',6'-Dmt-D-Arg-Phe-Lys-NH2 (SS-02). In another embodiment, the peptide
comprises a phenylalanine or a 2',6'-dimethylphenylalanine residue at the
N-terminus. For example, the peptide may have the formula
Phe-D-Arg-Phe-Lys-NH2 (SS-20) or 2',6'-Dmp-D-Arg-Phe-Lys-NH2. In a
particular embodiment, the aromatic-cationic peptide has the formula
D-Arg-2',6'-Dmt-Lys-Phe-NH2 (SS-31).

[0057] In a particular embodiment, R1, R2, R3, R4,
R5, R6, R7, R8, R9, R10, R11, and
R12 are all hydrogen; and n is 4. In another embodiment, R1,
R2, R3, R4, R5, R6, R7, R8, R9,
and R11 are all hydrogen; R8 and R12 are methyl; R10
is hydroxyl; and n is 4.

[0058] The aromatic-cationic peptides may be administered in a variety of
ways. In some embodiments, the peptides are administered orally,
topically, intranasally, intraperitoneally, intravenously, or
subcutaneously.

[0060] FIGS. 2A and 2B are graphs showing the levels of oxidatively
modified proteins in the diaphragm of control, MV, and mechanically
ventilated rats treated with the mitochondrial-targeted antioxidant SS-31
(MVSS). FIG. 2A shows the levels of 4-hydroxyl-nonenal-conjugated
proteins in the diaphragm of the three experimental groups. The image
above the histograph is a representative western blot of data from the
three experimental groups. FIG. 2B shows the levels of protein carbonyls
in the diaphragm of the three experimental groups. The image above the
histograph is a representative western blot of data from the three
experimental groups.

[0061] FIG. 3 is a graph demonstrating the effects of prolonged MV on the
diaphragmatic force-frequency response (in vitro) in control and
mechanically ventilated rats in the presence and absence of mitochondrial
targeted antioxidants.

[0062]FIG. 4 is a graph showing the fiber cross-sectional area (CSA) in
diaphragm muscle myofibers from control and mechanically ventilated rats
with (MVSS).

[0063] FIG. 5A-5C are graphs showing protease activity. FIG. 5A shows the
activity of the 20S proteasome. FIG. 5B shows the mRNA and protein levels
of atrogin-1. FIG. 5C shows the mRNA and protein levels of MuRF-1. The
images above the histograms in FIGS. 5B and 5C are representative western
blots of data from the three experimental groups.

[0064] FIGS. 6A and 6B are graphs of calpain 1 and caspase 3 activity in
the diaphragm from control and mechanically ventilated animals in the
presence and absence of mitochondrial-targeted antioxidants (MVSS). FIG.
6A shows the active form of calpain 1 in diaphragm muscle at the
completion of 12 hours of MV. FIG. 5B shows the cleaved and active band
of caspase-3 in diaphragm muscle at the completion of 12 hours of MV. The
images above the histograms are representative western blots of data from
the three experimental groups.

[0065] FIGS. 7A and 7B are graphs illustrating calpain and caspase-3
activity in the diaphragm from control and mechanically ventilated
animals in the presence and absence of a mitochondrial-targeted
antioxidants (MV). FIG. 7A shows levels of the 145 kDa
α-II-spectrin break-down product (SBPD) in diaphragm muscle
following 12 hours of MV. FIG. 7B shows the levels of the 120 kDa
α-II-spectrin break-down product (SBPD 120 kDa) in diaphragm muscle
following 12 hours of MV. The images above the histograms are
representative western blots of data from the three experimental groups.

[0066]FIG. 8 is a graph showing the ratio of actin to total sarcomeric
protein levels in the diaphragm from control and mechanically ventilated
animals in the presence and absence of mitochondrial-targeted
antioxidants (MV). The image above the histogram is a representative
western blot of data from the three experimental groups.

[0071] FIG. 13A-13D are graphs illustrating that casting for 7 days caused
significant decrease in weight of soleus muscle (FIG. 13A) which was
prevented by SS-31. Casting also significantly reduced mitochondrial
state 3 (FIG. 13C) respiration, but had no effect on state 4 (FIG. 13D),
thus resulting in a significant decrease in RCR (FIG. 13B). All of the
foregoing defects were prevented by SS-31.

[0072] FIGS. 14A and 14B are graphs showing that casting for 7 days
significantly increased H2O2 production by mitochondrial
isolated from soleus muscle, which was prevented by SS-31 (FIG. 14A).
FIG. 14B illustrates that SS-31 prevented the loss of cross sectional
area of all three types of fibers as shown.

[0073] FIG. 15A-15D are graphs showing that casting for 7 days increased
oxidative damage in soleus muscle, as measured by lipid peroxidation
(FIG. 15A), which was blocked by SS-31. Casting also significantly
increased protease activity of calpain-1 (FIG. 15B), caspase-3 (FIG. 15C)
and caspase-12 (FIG. 15D) in the soleus muscle, which was prevented by
SS-31.

[0074] FIG. 16A-16D are graphs showing that casting for 7 days reduced
plantaris weight (FIG. 16A) and mitochondrial RCR (FIG. 16B) in the
plantaris muscle, which was prevented by SS-31. FIG. 16C shows state 3
respiration, and FIG. 16D shows state 4 respiration.

[0075]FIG. 17 is a graph showing that casting for 7 days significantly
increased H2O2 production by mitochondrial isolated from
plantaris muscle, which was prevented by SS-31 (FIG. 17A). FIG. 17B
illustrates that SS-31 prevented the loss of cross sectional area of two
types of fibers as shown.

[0076] FIG. 18A-18D are graphs showing that casting for 7 days increased
oxidative damage in plantaris muscle, as measured by lipid peroxidation
(FIG. 18A), which was blocked by SS-31. Casting also increased protease
activity of calpain-1 (FIG. 18B), caspase-3 (FIG. 18C) and caspase-12
(FIG. 18D) in the plantaris muscle, which was prevented by SS-31.

DETAILED DESCRIPTION

[0077] It is to be appreciated that certain aspects, modes, embodiments,
variations and features of the invention are described below in various
levels of detail in order to provide a substantial understanding of the
present invention. The definitions of certain terms as used in this
specification are provided below. Unless defined otherwise, all technical
and scientific terms used herein generally have the same meaning as
commonly understood by one of ordinary skill in the art to which this
invention belongs.

[0079] As used in this specification and the appended claims, the singular
forms "a," "an" and "the" include plural referents unless the content
clearly dictates otherwise. For example, reference to "a peptide"
includes a combination of two or more peptides, and the like.

[0080] As used herein, phrases such as element A is "associated with"
element B mean both elements exist, but should not be interpreted as
meaning one element necessarily is causally linked to the other.

[0081] As used herein, the "administration" of an agent, drug, or peptide
to a subject includes any route of introducing or delivering to a subject
a compound to perform its intended function. Administration can be
carried out by any suitable route, including orally, intranasally,
parenterally (intravenously, intramuscularly, intraperitoneally, or
subcutaneously), or topically. Administration includes
self-administration and the administration by another.

[0082] As used herein, the term "amino acid" includes naturally-occurring
amino acids, L-amino acids, D-amino acids, and synthetic amino acids, as
well as amino acid analogs and amino acid mimetics that function in a
manner similar to the naturally-occurring amino acids.
Naturally-occurring amino acids are those encoded by the genetic code, as
well as those amino acids that are later modified, e.g., hydroxyproline,
γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers
to compounds that have the same basic chemical structure as a
naturally-occurring amino acid, e.g., an α-carbon that is bound to
a hydrogen, a carboxyl group, an amino group, and an R group, e.g.,
homoserine, norleucine, methionine sulfoxide, methionine methyl
sulfonium. Such analogs have modified R-groups (e.g., norleucine) or
modified peptide backbones, but retain the same basic chemical structure
as a naturally-occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in a
manner similar to a naturally-occurring amino acid. Amino acids can be
referred to herein by either their commonly known three letter symbols or
by the one-letter symbols recommended by the IUPAC-IUB Biochemical
Nomenclature Commission.

[0083] As used herein, the terms "effective amount" or "therapeutically
effective amount" or "pharmaceutically effective amount" refer to a
quantity sufficient to achieve a desired therapeutic and/or prophylactic
effect, e.g., an amount which results in the prevention of, or a decrease
in, muscle dysfunction or atrophy or one or more symptoms associated
therewith. In the context of therapeutic or prophylactic applications,
the amount of a composition administered to the subject will depend on
the type and severity of the disease and on the characteristics of the
individual, such as general health, age, sex, body weight and tolerance
to drugs. It will also depend on the degree, severity and type of
disease. The skilled artisan will be able to determine appropriate
dosages depending on these and other factors. The compositions can also
be administered in combination with one or more additional therapeutic
compounds. In the methods described herein, the aromatic-cationic
peptides may be administered to a subject having one or more signs or
symptoms of the effect associated with muscle disuse, MV implementation,
and the like. For example, a "therapeutically effective amount" of one or
more aromatic-cationic peptides refers to an amount sufficient to, at a
minimum, ameliorate MV-induced or disuse-induced muscle atrophy,
dysfunction, degradation, contractile dysfunction, damage, etc.

[0084] As used herein, the term "medical condition" includes, but is not
limited to, any condition or disease manifested as one or more physical
and/or psychological symptoms for which treatment and/or prevention is
desirable, and includes previously and newly identified diseases and
other disorders. For example, a medical condition may be MV-induced or
disuse-induced skeletal muscle atrophy or dysfunction or contractile
dysfunction or any associated symptoms or complications.

[0085] An "isolated" or "purified" polypeptide or peptide is substantially
free of cellular material or other contaminating polypeptides from the
cell or tissue source from which the agent is derived, or substantially
free from chemical precursors or other chemicals when chemically
synthesized. For example, an isolated aromatic-cationic peptide would be
free of materials that would interfere with diagnostic or therapeutic
uses of the agent. Such interfering materials may include enzymes,
hormones and other proteinaceous and nonproteinaceous solutes.

[0086] As used herein, the term "net charge" refers to the balance of the
number of positive charges and the number of negative charges carried by
the amino acids present in the peptide. In this specification, it is
understood that net charges are measured at physiological pH. The
naturally occurring amino acids that are positively charged at
physiological pH include L-lysine, L-arginine, and L-histidine. The
naturally occurring amino acids that are negatively charged at
physiological pH include L-aspartic acid and L-glutamic acid.

[0087] As used herein, the terms "polypeptide," "peptide," and "protein"
are used interchangeably herein to mean a polymer comprising two or more
amino acids joined to each other by peptide bonds or modified peptide
bonds, i.e., peptide isosteres. Polypeptide refers to both short chains,
commonly referred to as peptides, glycopeptides or oligomers, and to
longer chains, generally referred to as proteins. Polypeptides may
contain amino acids other than the 20 gene-encoded amino acids.
Polypeptides include amino acid sequences modified either by natural
processes, such as post-translational processing, or by chemical
modification techniques that are well known in the art.

[0088] As used herein, "prevention" or "preventing" of a disorder or
condition refers to a compound that, in a statistical sample, reduces the
occurrence of the disorder or condition in the treated sample relative to
an untreated control sample, or delays the onset or reduces the severity
of one or more symptoms of the disorder or condition relative to the
untreated control sample. As used herein, preventing skeletal muscle
dysfunction includes preventing the initiation of skeletal muscle
dysfunction, delaying the initiation of skeletal muscle dysfunction,
preventing the progression or advancement of skeletal muscle dysfunction,
slowing the progression or advancement of skeletal muscle dysfunction,
delaying the progression or advancement of skeletal muscle dysfunction,
and reversing the progression of skeletal muscle dysfunction from an
advanced to a less advanced stage.

[0089] As used herein, the terms "prolonged" or "prolonged-MV" or
"prolonged-disuse" in reference to the cause or correlation with muscle
weakness or muscle dysfunction or muscle atrophy, includes a time from at
least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, or 100
hours, to from at least about 1, 10, 20, 50, 75, 100 or greater hours,
days, or years.

[0090] As used herein, the term "simultaneous" therapeutic use refers to
the administration of at least two active ingredients by the same route
and at the same time or at substantially the same time.

[0091] As used herein, the term "separate" therapeutic use refers to an
administration of at least two active ingredients at the same time or at
substantially the same time by different routes.

[0092] The term "overlapping" therapeutic use refers to administration of
one or more active ingredients at different but overlapping times.
Overlapping therapeutic use includes administration of active ingredients
by different routes or by the same route.

[0093] As used herein, the term "sequential" therapeutic use refers to
administration of at least two active ingredients at different times, the
administration route being identical or different. More particularly,
sequential use refers to the whole administration of one of the active
ingredients before administration of the other or others commences. It is
thus possible to administer one of the active ingredients over several
minutes, hours, or days before administering the other active ingredient
or ingredients. There is no simultaneous treatment in this case.

[0094] As used herein, the term "subject" refers to a member of any
vertebrate species. The methods of the presently disclosed subject matter
are particularly useful for warm-blooded vertebrates. Provided herein is
the treatment of mammals such as humans, as well as those mammals of
importance due to being endangered, of economic importance (animals
raised on farms for consumption by humans) and/or social importance
(animals kept as pets or in zoos) to humans. In particular embodiments,
the subject is a human.

[0095] As used herein, the term "muscle infirmity" refers to reduced or
aberrant muscle function and includes, for example, one or more of muscle
weakness, muscle dysfunction, atrophy, disuse, degradation, contractile
dysfunction or damage. One example of muscle infirmity is mechanical
ventilation (MV)-induced diaphragm weakness. Another example of muscle
infirmity is muscle weakness induced by muscle disuse, such as by casting
a limb. Muscle infirmity can be induced, derived or develop for one or
more of several reasons, including but not limited to age, genetics,
disease (e.g., infection), mechanical or chemical causes. Some
non-limiting examples in which muscle infirmity arises include aging,
prolonged bed rest, muscle weakness associated with microgravity (e.g.,
as in space flight), drug induced muscle weakness (e.g., as an effect of
statins, antiretrovirals and thiazolidinediones), and cachexia due to
cancer or other diseases. In some instances, muscle infirmity, such as
skeletal muscle infirmity, results from oxidative stress caused by the
production of reactive oxygen species ("ROS") by enzymes (e.g., xanthine
oxidase, NADPH oxidase) and/or the mitochondria within the muscle cells
themselves. Such ROS may be produced under any number of circumstances,
including those listed above. Muscle infirmity or the extent of muscle
infirmity can be determined by evaluating one more physical and/or
physiological parameters.

[0096] As used herein, the terms "treating" or "treatment" or
"alleviation" refers to therapeutic treatment, wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. A subject is successfully "treated" for MV-induced or
disuse-induced muscle infirmity, if after receiving a therapeutic amount
of the aromatic-cationic peptides according to the methods described
herein, the subject shows observable and/or measurable reduction in or
absence of one or more signs and symptoms of MV-induced or disuse-induced
infirmity, such as, e.g., MV-induced or disuse-induced muscle atrophy,
dysfunction, degradation, contractile dysfunction, damage, and the like.
It is also to be appreciated that the various modes of treatment or
prevention of medical conditions as described are intended to mean
"substantial," which includes total but also less than total treatment or
prevention, and wherein some biologically or medically relevant result is
achieved. Treating muscle infirmity, as used herein, also refers to
treating any one or more of muscle dysfunction, atrophy, disuse,
degradation, contractile dysfunction, damage, etc.

I. Aromatic-Cationic Peptides

[0097] In one aspect, compositions and methods for the treatment or
prevention of skeletal muscle infirmity (e.g., weakness, atrophy,
dysfunction, etc.) are provided. In some embodiments, the compositions
and methods include administration of certain aromatic-cationic peptides,
or a pharmaceutically acceptable salt thereof, such as acetate salt or
trifluoroacetate salt. The aromatic-cationic peptides are water-soluble
and highly polar. Despite these properties, the peptides can readily
penetrate cell membranes. The aromatic-cationic peptides typically
include a minimum of three amino acids or a minimum of four amino acids,
covalently joined by peptide bonds. The maximum number of amino acids
present in the aromatic-cationic peptides is about twenty amino acids
covalently joined by peptide bonds. Suitably, the maximum number of amino
acids is about twelve, more preferably about nine, and most preferably
about six.

[0098] The amino acids of the aromatic-cationic peptides can be any amino
acid. As used herein, the term "amino acid" is used to refer to any
organic molecule that contains at least one amino group and at least one
carboxyl group. Typically, at least one amino group is at the α
position relative to a carboxyl group. The amino acids may be naturally
occurring. Naturally occurring amino acids include, for example, the
twenty most common levorotatory (L) amino acids normally found in
mammalian proteins, i.e., alanine (Ala), arginine (Arg), asparagine
(Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic
acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine
(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline
(Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr),
and valine (Val). Other naturally occurring amino acids include, for
example, amino acids that are synthesized in metabolic processes not
associated with protein synthesis. For example, the amino acids ornithine
and citrulline are synthesized in mammalian metabolism during the
production of urea. Another example of a naturally occurring amino acid
includes hydroxyproline (Hyp).

[0099] The peptides optionally contain one or more non-naturally occurring
amino acids. In some embodiments, the peptide has no amino acids that are
naturally occurring. The non-naturally occurring amino acids may be
levorotary (L-), dextrorotatory (D-), or mixtures thereof. Non-naturally
occurring amino acids are those amino acids that typically are not
synthesized in normal metabolic processes in living organisms, and do not
naturally occur in proteins. In addition, the non-naturally occurring
amino acids suitably are also not recognized by common proteases. The
non-naturally occurring amino acid can be present at any position in the
peptide. For example, the non-naturally occurring amino acid can be at
the N-terminus, the C-terminus, or at any position between the N-terminus
and the C-terminus. Pharmaceutically acceptable salts forms of the
peptides of the present technology are useful in the methods provided by
the present technology as described herein (e.g., but not limited to,
acetate salts or trifluoroacetate salts thereof).

[0100] The non-natural amino acids may, for example, comprise alkyl, aryl,
or alkylaryl groups not found in natural amino acids. Some examples of
non-natural alkyl amino acids include α-aminobutyric acid,
β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric
acid, and ε-aminocaproic acid. Some examples of non-natural aryl
amino acids include ortho, meta, and para-aminobenzoic acid. Some
examples of non-natural alkylaryl amino acids include ortho-, meta-, and
para-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.
Non-naturally occurring amino acids include derivatives of naturally
occurring amino acids. The derivatives of naturally occurring amino acids
may, for example, include the addition of one or more chemical groups to
the naturally occurring amino acid.

[0101] For example, one or more chemical groups can be added to one or
more of the 2', 3', 4', 5', or 6' position of the aromatic ring of a
phenylalanine or tyrosine residue, or the 4', 5', 6', or 7' position of
the benzo ring of a tryptophan residue. The group can be any chemical
group that can be added to an aromatic ring. Some examples of such groups
include branched or unbranched C1-C4 alkyl, such as methyl,
ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C1-C4
alkyloxy (i.e., alkoxy), amino, C1-C4 alkylamino and
C1-C4 dialkylamino (e.g., methylamino, dimethylamino), nitro,
hydroxyl, halo (i.e., fluoro, chloro, bromo, or iodo). Some specific
examples of non-naturally occurring derivatives of naturally occurring
amino acids include norvaline (Nva) and norleucine (Nle).

[0102] Another example of a modification of an amino acid in a peptide is
the derivatization of a carboxyl group of an aspartic acid or a glutamic
acid residue of the peptide. One example of derivatization is amidation
with ammonia or with a primary or secondary amine, e.g. methylamine,
ethylamine, dimethylamine or diethylamine. Another example of
derivatization includes esterification with, for example, methyl or ethyl
alcohol. Another such modification includes derivatization of an amino
group of a lysine, arginine, or histidine residue. For example, such
amino groups can be acylated. Some suitable acyl groups include, for
example, a benzoyl group or an alkanoyl group comprising any of the
C1-C4 alkyl groups mentioned above, such as an acetyl or
propionyl group.

[0103] The non-naturally occurring amino acids are suitably resistant or
insensitive to common proteases. Examples of non-naturally occurring
amino acids that are resistant or insensitive to proteases include the
dextrorotatory (D-) form of any of the above-mentioned naturally
occurring L-amino acids, as well as L- and/or D-non-naturally occurring
amino acids. The D-amino acids do not normally occur in proteins,
although they are found in certain peptide antibiotics that are
synthesized by means other than the normal ribosomal protein synthetic
machinery of the cell. As used herein, the D-amino acids are considered
to be non-naturally occurring amino acids.

[0104] In order to minimize protease sensitivity, the peptides should have
less than five, preferably less than four, more preferably less than
three, and most preferably, less than two contiguous L-amino acids
recognized by common proteases, irrespective of whether the amino acids
are naturally or non-naturally occurring. Optimally, the peptide has only
D-amino acids, and no L-amino acids. If the peptide contains protease
sensitive sequences of amino acids, at least one of the amino acids is
preferably a non-naturally-occurring D-amino acid, thereby conferring
protease resistance. An example of a protease sensitive sequence includes
two or more contiguous basic amino acids that are readily cleaved by
common proteases, such as endopeptidases and trypsin. Examples of basic
amino acids include arginine, lysine and histidine.

[0105] The aromatic-cationic peptides should have a minimum number of net
positive charges at physiological pH in comparison to the total number of
amino acid residues in the peptide. The minimum number of net positive
charges at physiological pH will be referred to below as (pm). The
total number of amino acid residues in the peptide will be referred to
below as (r). The minimum number of net positive charges discussed below
are all at physiological pH. The term "physiological pH" as used herein
refers to the normal pH in the cells of the tissues and organs of the
mammalian body. For instance, the physiological pH of a human is normally
approximately 7.4, but normal physiological pH in mammals may be any pH
from about 7.0 to about 7.8.

[0106] Typically, a peptide has a positively charged N-terminal amino
group and a negatively charged C-terminal carboxyl group. The charges
cancel each other out at physiological pH. As an example of calculating
net charge, the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one
negatively charged amino acid (i.e., Glu) and four positively charged
amino acids (i.e., two Arg residues, one Lys, and one His). Therefore,
the above peptide has a net positive charge of three.

[0107] In one embodiment, the aromatic-cationic peptides have a
relationship between the minimum number of net positive charges at
physiological pH (pm) and the total number of amino acid residues
(r) wherein 3pm is the largest number that is less than or equal to
r+1. In this embodiment, the relationship between the minimum number of
net positive charges (pm) and the total number of amino acid
residues (r) is as follows:

[0108] In another embodiment, the aromatic-cationic peptides have a
relationship between the minimum number of net positive charges (pm)
and the total number of amino acid residues (r) wherein 2pm is the
largest number that is less than or equal to r+1. In this embodiment, the
relationship between the minimum number of net positive charges (pm)
and the total number of amino acid residues (r) is as follows:

[0109] In one embodiment, the minimum number of net positive charges
(pm) and the total number of amino acid residues (r) are equal. In
another embodiment, the peptides have three or four amino acid residues
and a minimum of one net positive charge, suitably, a minimum of two net
positive charges and more preferably a minimum of three net positive
charges.

[0110] It is also important that the aromatic-cationic peptides have a
minimum number of aromatic groups in comparison to the total number of
net positive charges (pt). The minimum number of aromatic groups
will be referred to below as (a). Naturally occurring amino acids that
have an aromatic group include the amino acids histidine, tryptophan,
tyrosine, and phenylalanine. For example, the hexapeptide
Lys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributed
by the lysine and arginine residues) and three aromatic groups
(contributed by tyrosine, phenylalanine and tryptophan residues).

[0111] The aromatic-cationic peptides should also have a relationship
between the minimum number of aromatic groups (a) and the total number of
net positive charges at physiological pH (pt) wherein 3a is the
largest number that is less than or equal to pt+1, except that when
pt is 1, a may also be 1. In this embodiment, the relationship
between the minimum number of aromatic groups (a) and the total number of
net positive charges (pt) is as follows:

[0112] In another embodiment, the aromatic-cationic peptides have a
relationship between the minimum number of aromatic groups (a) and the
total number of net positive charges (pt) wherein 2a is the largest
number that is less than or equal to pt+1. In this embodiment, the
relationship between the minimum number of aromatic amino acid residues
(a) and the total number of net positive charges (pt) is as follows:

[0113] In another embodiment, the number of aromatic groups (a) and the
total number of net positive charges (pt) are equal.

[0114] Carboxyl groups, especially the terminal carboxyl group of a
C-terminal amino acid, are suitably amidated with, for example, ammonia
to form the C-terminal amide. Alternatively, the terminal carboxyl group
of the C-terminal amino acid may be amidated with any primary or
secondary amine. The primary or secondary amine may, for example, be an
alkyl, especially a branched or unbranched C1-C4 alkyl, or an
aryl amine. Accordingly, the amino acid at the C-terminus of the peptide
may be converted to an amido, N-methylamido, N-ethylamido,
N,N-dimethylamido, N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido
or N-phenyl-N-ethylamido group. The free carboxylate groups of the
asparagine, glutamine, aspartic acid, and glutamic acid residues not
occurring at the C-terminus of the aromatic-cationic peptides may also be
amidated wherever they occur within the peptide. The amidation at these
internal positions may be with ammonia or any of the primary or secondary
amines described above.

[0115] In one embodiment, the aromatic-cationic peptide is a tripeptide
having two net positive charges and at least one aromatic amino acid. In
a particular embodiment, the aromatic-cationic peptide is a tripeptide
having two net positive charges and two aromatic amino acids.

[0116] Aromatic-cationic peptides include, but are not limited to, the
following peptide examples:

[0117] In one embodiment, the peptides have mu-opioid receptor agonist
activity (i.e., they activate the mu-opioid receptor). Peptides which
have mu-opioid receptor agonist activity are typically those peptides
which have a tyrosine residue or a tyrosine derivative at the N-terminus
(i.e., the first amino acid position). Suitable derivatives of tyrosine
include 2'-methyltyrosine (Mmt); 2',6'-dimethyltyrosine (2'6'-Dmt);
3',5'-dimethyltyrosine (3'5'Dmt); N,2',6'-trimethyltyrosine (Tmt); and
2'-hydroxy-6'-methyltryosine (Hmt).

[0118] In one embodiment, a peptide that has mu-opioid receptor agonist
activity has the formula Tyr-D-Arg-Phe-Lys-NH2 (referred to herein
as "SS-01"). SS-01 has a net positive charge of three, contributed by the
amino acids tyrosine, arginine, and lysine and has two aromatic groups
contributed by the amino acids phenylalanine and tyrosine. The tyrosine
of SS-01 can be a modified derivative of tyrosine such as in
2',6'-dimethyltyrosine to produce the compound having the formula
2',6'-Dmt-D-Arg-Phe-Lys-NH2 (referred to herein as "SS--O2").
SS-02 has a molecular weight of 640 and carries a net three positive
charge at physiological pH. SS-02 readily penetrates the plasma membrane
of several mammalian cell types in an energy-independent manner (Zhao et
al., J. Pharmacol Exp Ther., 304:425-432, 2003).

[0119] Alternatively, in other instances, the aromatic-cationic peptide
does not have mu-opioid receptor agonist activity. For example, during
long-term treatment, such as in a chronic disease state or condition, the
use of an aromatic-cationic peptide that activates the mu-opioid receptor
may be contraindicated. In these instances, the potentially adverse or
addictive effects of the aromatic-cationic peptide may preclude the use
of an aromatic-cationic peptide that activates the mu-opioid receptor in
the treatment regimen of a human patient or other mammal. Potential
adverse effects may include sedation, constipation and respiratory
depression. In such instances an aromatic-cationic peptide that does not
activate the mu-opioid receptor may be an appropriate treatment. Peptides
that do not have mu-opioid receptor agonist activity generally do not
have a tyrosine residue or a derivative of tyrosine at the N-terminus
(i.e., amino acid position 1). The amino acid at the N-terminus can be
any naturally occurring or non-naturally occurring amino acid other than
tyrosine. In one embodiment, the amino acid at the N-terminus is
phenylalanine or its derivative. Exemplary derivatives of phenylalanine
include 2'-methylphenylalanine (Mmp), 2',6'-dimethylphenylalanine
(2',6'-Dmp), N,2',6'-trimethylphenylalanine (Tmp), and
2'-hydroxy-6'-methylphenylalanine (Hmp).

[0120] An example of an aromatic-cationic peptide that does not have
mu-opioid receptor agonist activity has the formula
Phe-D-Arg-Phe-Lys-NH2 (referred to herein as "SS-20").
Alternatively, the N-terminal phenylalanine can be a derivative of
phenylalanine such as 2',6'-dimethylphenylalanine (2'6'-Dmp). SS-01
containing 2',6'-dimethylphenylalanine at amino acid position 1 has the
formula 2',6'-Dmp-D-Arg-Phe-Lys-NH2. In one embodiment, the amino
acid sequence of SS-02 is rearranged such that Dmt is not at the
N-terminus. An example of such an aromatic-cationic peptide that does not
have mu-opioid receptor agonist activity has the formula
D-Arg-2'6'-Dmt-Lys-Phe-NH2.

[0121] Suitable substitution variants of the peptides listed herein
include conservative amino acid substitutions. Amino acids may be grouped
according to their physicochemical characteristics as follows:

[0127] Substitutions of an amino acid in a peptide by another amino acid
in the same group is referred to as a conservative substitution and may
preserve the physicochemical characteristics of the original peptide. In
contrast, substitutions of an amino acid in a peptide by another amino
acid in a different group is generally more likely to alter the
characteristics of the original peptide.

[0128] Examples of peptides that activate mu-opioid receptors include, but
are not limited to, the aromatic-cationic peptides shown in Table 5.

[0130] The amino acids of the peptides shown in Table 5 and 6 may be in
either the L- or the D-configuration.

[0131] The peptides may be synthesized by any of the methods well known in
the art. Suitable methods for chemically synthesizing the protein
include, for example, those described by Stuart and Young in Solid Phase
Peptide Synthesis, Second Edition, Pierce Chemical Company (1984), and in
Methods Enzymol., 289, Academic Press, Inc, New York (1997).

II. Use of Aromatic-Cationic Peptides

[0132] Elevated ROS emissions have been shown to be a causative agent for
oxidative stress and the concomitant muscle infirmities (e.g., weakness,
atrophy, dysfunction) in MV-induced and disuse-induced skeletal muscle
weakness. Mitochondria in the muscle cells appear to be the leading ROS
producers, and as shown below in the Experimental Examples, mitochondrial
ROS emissions play a role in MV-induced and disuse-induced oxidative
stress that leads to skeletal muscle (e.g., diaphragm, soleus and
plantaris muscle) infirmities. While NADPH activation and xanthine
oxidase activation also play a role in ROS production, NADPH activity is
minimal (i.e. 5%) and inhibition of xanthine oxidase activity does not
completely protect against the effects of skeletal muscle disuse-induced
or MV-induced oxidative stress and the concomitant atrophy and weakness.
Moreover, mitochondrial ROS emission is an up-stream signal for the MV-
or disuse-induced activation of proteases, e.g., calpain, caspase-3
and/or caspase-12, in the diaphragm and other skeletal muscles.

[0133] Accordingly, the present disclosure describes methods and
compositions including mitochondria-targeted, antioxidant,
aromatic-cationic peptides capable of reducing mitochondrial ROS
production in the diaphragm during prolonged MV, or in other skeletal
muscles, e.g., soleus or plantaris muscle, during limb immobilization or
muscle disuse in general.

[0134] In one aspect, the present disclosure provides a
mitochondria-targeted antioxidant, i.e., D-Arg-2',6'Dmt-Lys-Phe-NH2
or "SS-31" or a pharmaceutically acceptable salt thereof, such as acetate
salt or trifluoroacetate salt. For example, in some embodiments, SS-31 is
used as a therapeutic and/or a prophylactic agent in subjects suffering
from, or at risk of suffering from muscle infirmities such as weakness,
atrophy, dysfunction, etc. caused by mitochondrial derived ROS. In some
embodiments, SS-31 decreases mitochondrial ROS emission in muscle.
Additionally or alternatively, in some embodiments, SS-31 selectively
concentrates in the mitochondria of skeletal muscle and provides radical
scavenging of H2O2, OH--, and ONOO--, and in some embodiments,
radical scavenging is on a dose-dependent basis.

[0135] In some embodiments, methods of treating muscle infirmities (e.g.,
weakness, atrophy, dysfunction, etc.) are described. In such therapeutic
applications, compositions or medicaments including an aromatic cationic
peptide such as SS-31 or a pharmaceutically acceptable salt thereof, such
as acetate salt or trifluoroacetate salt, are administered to a subject
suspected of, or already suffering from, muscle infirmity, in an amount
sufficient to prevent, reduce, alleviate, or at least partially arrest,
the symptoms of muscle infirmity, including its complications and
intermediate pathological phenotypes in development of the infirmity. As
such, the invention provides methods of treating an individual afflicted,
or suspected of suffering from muscle infirmities described herein. In
one embodiment, the aromatic cationic peptide SS-31, or a
pharmaceutically acceptable salt thereof, such as acetate salt or
trifluoroacetate salt, is administered.

[0136] In another aspect, the disclosure provides a method for preventing,
or reducing the likelihood of muscle infirmity, as described herein, by
administering to the subject an aromatic-cationic peptide that prevents
or reduces the likelihood of the initiation or progression of the
infirmity. Subjects at risk for developing muscle infirmity can be
readily identified, e.g., a subject preparing for or about to undergo MV
or related diaphragmatic muscles disuse or any other skeletal muscle
disuse that may be envisaged by a medical professional (e.g., casting a
limb). In one embodiment, the aromatic cationic peptide includes SS-31 or
a pharmaceutically acceptable salt thereof, such as acetate salt or
trifluoroacetate salt.

[0137] In such prophylactic applications, a pharmaceutical composition or
medicament comprising one or more aromatic-cationic peptides or a
pharmaceutically acceptable salt thereof, such as acetate salt or
trifluoroacetate salt, is administered to a subject susceptible to, or
otherwise at risk of muscle infirmity in an amount sufficient to
eliminate or reduce the risk, lessen the severity, or delay the onset of
muscle infirmity, including biochemical, histologic and/or behavioral
symptoms of the infirmity, its complications and intermediate
pathological phenotypes presenting during development of the infirmity.
Administration of one or more of the aromatic-cationic peptide disclosed
herein can occur prior to the manifestation of symptoms characteristic of
the aberrancy, such that the disorder is prevented or, alternatively,
delayed in its progression. The appropriate compound can be determined
based on screening assays described above or as well known in the art. In
one embodiment, the pharmaceutical composition includes SS-31 or a
pharmaceutically acceptable salt thereof, such as acetate salt or
trifluoroacetate salt.

[0138] In various embodiments, suitable in vitro or in vivo assays are
performed to determine the effect of a specific aromatic-cationic
peptide-based therapeutic and whether its administration is indicated for
treatment. In various embodiments, assays can be performed with
representative animal models, to determine if a given aromatic-cationic
peptide-based therapeutic exerts the desired effect in preventing or
treating muscle weakness (e.g., atrophy, dysfunction, etc.). Compounds
for use in therapy can be tested in suitable animal model systems
including, but not limited to rats, mice, chicken, cows, monkeys,
rabbits, and the like, prior to testing in human subjects. Similarly, for
in vivo testing, any of the animal model system known in the art can be
used prior to administration to human subjects.

[0139] In some embodiments, subjects in need of protection from or
treatment of muscle infirmity also include subjects suffering from a
disease, condition or treatment associated with oxidative damage.
Typically, the oxidative damage is caused by free radicals, such as
reactive oxygen species (ROS) and/or reactive nitrogen species (RNS).
Examples of ROS and RNS include hydroxyl radical (HO.), superoxide anion
radical (O2.sup.-), nitric oxide (NO.), hydrogen peroxide
(H2O2), hypochlorous acid (HOCl) and peroxynitrite anion
(ONOO.sup.-).

[0140] Respiratory muscle infirmity may result from prolonged MV, e.g.,
greater than 12 hours. In some embodiments, the respiratory muscle
infirmity is due to contractile dysfunction and/or atrophy. However, such
prolonged MV is not limited to any specific time-length. For example, in
some embodiments, prolonged MV includes a time from at least about 0.5,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, or 100 hours, to from at
least about 1, 10, 20, 50, 75, 100 or greater hours, days, or years. In
another embodiment, prolonged MV includes a time from at least about 5,
6, 7, 8, 9 or 10 hours, to from at least about 10, 20 or 50 hours. In
some embodiments, prolonged MV is from about at least 10-12 hours to any
time greater than the 10-12 hour period. In some embodiments,
administration of the aromatic peptide compositions described herein is
provided at any time during MV or muscle immobilization. In some
embodiments, one or more doses of a cationic peptide composition is
administered before MV, immediately after MV initiation, during MV,
and/or immediately after MV.

[0141] Muscle disuse atrophy also presents an obstacle to recovery for
subjects attempting to reestablishment muscle function subsequent to
immobilization. In this respect, the aromatic-cationic peptides or a
pharmaceutically acceptable salt thereof, such as acetate salt or
trifluoroacetate salt, described herein provide for prophylactic and
therapeutic methods of treating a subject having or at risk of having
skeletal muscle-associated infirmities. Such muscle infirmities result
from or include, but are not limited to, muscle disuse or MV, wherein the
muscle disuse or MV induces apoptosis, oxidative stress, oxidative
damage, contractile dysfunction, muscle atrophy, muscle proteolysis,
protease activation, mitochondrial-derived ROS emission, mitochondrial
H2O2 release, mitochondrial uncoupling, impaired mitochondria
coupling, impaired state 3 mitochondrial respiration, impaired state 4
mitochondrial respiration, decreased respiratory control ration (RCR),
reduced lipid peroxidation, or any combination thereof.

[0142] Composition comprising a cationic peptide disclosed herein to treat
or prevent muscle infirmity associated with muscle immobilization e.g.,
due to casting or other disuse can be administered at any time before,
during or after the immobilization or disuse. For example, in some
embodiments, one or more doses of a cationic peptide composition is
administered before muscle immobilization or disuse, immediately after
muscle immobilization or disuse, during the course of muscle
immobilization or disuse, and/or after muscle immobilization or disuse
(e.g., after cast removal). By way of example, and not by way of
limitation, in some embodiments, a cationic peptide (e.g., SS-31 or a
pharmaceutically acceptable salt thereof, such as acetate salt or
trifluoroacetate salt) is administered once per day, twice per day, three
times per day, four times per day six times per day or more, for the
duration of the immobilization or disuse. In other embodiments, a
cationic peptide (e.g., SS-31 or a pharmaceutically acceptable salt
thereof, such as acetate salt or trifluoroacetate salt) is administered
daily, every other day, twice, three times, or for times per week, or
once, twice three, four, five or six times per month for the duration of
the immobilization or disuse.

[0143] In some embodiment, methods to treat or prevent muscle infirmity
due to muscle disuse or disuse atrophy, associated with loss of muscle
mass and strength, are also disclosed. Atrophy is a physiological process
relating to the reabsorption and degradation of tissues, e.g., fibrous
muscle tissue, which involves apoptosis at the cellular level. When
atrophy occurs from loss of trophic support or other disease, it is known
as pathological atrophy. Such atrophy or pathological atrophy may result
from, or is related to, limb immobilization, prolonged limb
immobilization, casting limb immobilization, MV, prolonged MV, extended
bed rest cachexia, congestive heart failure, liver disease, sarcopenia,
wasting, poor nourishment, poor circulation, hormonal irregularities,
loss of nerve function, and the like. Accordingly, the present methods
provide for the prevention and/or treatment of muscle infirmities,
including skeletal muscle atrophy, in a subject by administering an
effective amount of an aromatic-cationic peptide or a pharmaceutically
acceptable salt thereof, such as acetate salt or trifluoroacetate salt to
a subject in need thereof.

[0144] Additional examples of muscle infirmitites which can be treated,
prevented, or alleviated by administering the compositions and
formulations disclosed herein include, without limitation, age-related
muscle infirmities, muscle infirmities associated with prolonged bed
rest, muscle infirmities such as weakness and atrophy associated with
microgravity, as in space flight, muscle infirmities associated with
effects of certain drugs (e.g., statins, antiretrovirals, and
thiazolidinediones (TZDs)), and muscle infirmities such as cachexia, for
example cachexia caused by cancer or other diseases.

III. Modes of Administration and Dosages

[0145] Any method known to those in the art for contacting a cell, organ
or tissue with a peptide may be employed. Suitable methods include in
vitro, ex vivo, or in vivo methods. In vivo methods typically include the
administration of an aromatic-cationic peptide, such as those described
above, to a mammal, suitably a human. When used in vivo for therapy, the
aromatic-cationic peptides or a pharmaceutically acceptable salt thereof,
such as acetate salt or trifluoroacetate salt are administered to the
subject in effective amounts (i.e., amounts that have desired therapeutic
effect). The dose and dosage regimen will depend upon the degree of the
muscle infirmity in the subject, the characteristics of the particular
aromatic-cationic peptide used, e.g., its therapeutic index, the subject,
and the subject's history.

[0146] The effective amount may be determined during pre-clinical trials
and clinical trials by methods familiar to physicians and clinicians. An
effective amount of a peptide useful in the methods may be administered
to a mammal in need thereof by any of a number of well-known methods for
administering pharmaceutical compounds. The peptide may be administered
systemically or locally.

[0147] The peptide may be formulated as a pharmaceutically acceptable
salt. The term "pharmaceutically acceptable salt" means a salt prepared
from a base or an acid which is acceptable for administration to a
patient, such as a mammal (e.g., salts having acceptable mammalian safety
for a given dosage regime). However, it is understood that the salts are
not required to be pharmaceutically acceptable salts, such as salts of
intermediate compounds that are not intended for administration to a
patient. Pharmaceutically acceptable salts can be derived from
pharmaceutically acceptable inorganic or organic bases and from
pharmaceutically acceptable inorganic or organic acids. In addition, when
a peptide contains both a basic moiety, such as an amine, pyridine or
imidazole, and an acidic moiety such as a carboxylic acid or tetrazole,
zwitterions may be formed and are included within the term "salt" as used
herein. Salts derived from pharmaceutically acceptable inorganic bases
include ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,
manganic, manganous, potassium, sodium, and zinc salts, and the like.
Salts derived from pharmaceutically acceptable organic bases include
salts of primary, secondary and tertiary amines, including substituted
amines, cyclic amines, naturally-occurring amines and the like, such as
arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine,
diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,
ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,
glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine,
methylglucamine, morpholine, piperazine, piperadine, polyamine resins,
procaine, purines, theobromine, triethylamine, trimethylamine,
tripropylamine, tromethamine and the like. Salts derived from
pharmaceutically acceptable inorganic acids include salts of boric,
carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric or
hydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Salts
derived from pharmaceutically acceptable organic acids include salts of
aliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic,
lactobionic, malic, and tartaric acids), aliphatic monocarboxylic acids
(e.g., acetic, butyric, formic, propionic and trifluoroacetic acids),
amino acids (e.g., aspartic and glutamic acids), aromatic carboxylic
acids (e.g., benzoic, p-chlorobenzoic, diphenylacetic, gentisic,
hippuric, and triphenylacetic acids), aromatic hydroxyl acids (e.g.,
o-hydroxybenzoic, p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and
3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids
(e.g., fumaric, maleic, oxalic and succinic acids), glucuronic, mandelic,
mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids (e.g.,
benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic, isethionic,
methanesulfonic, naphthalenesulfonic, naphthalene-1,5-disulfonic,
naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,
and the like. In some embodiments, a pharmaceutically acceptable salt
includes acetate salt or trifluoroacetate salt.

[0148] The aromatic-cationic peptides or a pharmaceutically acceptable
salt thereof, such as acetate salt or trifluoroacetate salt, described
herein can be incorporated into pharmaceutical compositions for
administration, singly or in combination, to a subject for the treatment
or prevention of a disorder described herein. Such compositions typically
include the active agent and a pharmaceutically acceptable carrier. As
used herein the term "pharmaceutically acceptable carrier" includes
saline, solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the like,
compatible with pharmaceutical administration. Supplementary active
compounds can also be incorporated into the compositions.

[0149] Pharmaceutical compositions are typically formulated to be
compatible with its intended route of administration. Examples of routes
of administration include parenteral (e.g., intravenous, intradermal,
intraperitoneal or subcutaneous), oral, inhalation, transdermal
(topical), intraocular, iontophoretic, and transmucosal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a sterile
diluent such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other synthetic
solvents; antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating agents
such as ethylenediaminetetraacetic acid; buffers such as acetates,
citrates or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or bases, such
as hydrochloric acid or sodium hydroxide. The parenteral preparation can
be enclosed in ampoules, disposable syringes or multiple dose vials made
of glass or plastic. For convenience of the patient or treating
physician, the dosing formulation can be provided in a kit containing all
necessary equipment (e.g., vials of drug, vials of diluent, syringes and
needles) for a treatment course (e.g., 7 days of treatment).

[0150] Pharmaceutical compositions suitable for injectable use can include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. For intravenous administration, suitable
carriers include physiological saline, bacteriostatic water, Cremophor
EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In
all cases, a composition for parenteral administration must be sterile
and should be fluid to the extent that easy syringability exists. It
should be stable under the conditions of manufacture and storage and must
be preserved against the contaminating action of microorganisms such as
bacteria and fungi.

[0151] The aromatic-cationic peptide compositions can include a carrier,
which can be a solvent or dispersion medium containing, for example,
water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required particle
size in the case of dispersion and by the use of surfactants. Prevention
of the action of microorganisms can be achieved by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thiomerasol, and the like. Glutathione and other
antioxidants can be included to prevent oxidation. In many cases, it will
be preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions can be
brought about by including in the composition an agent which delays
absorption, for example, aluminum monostearate or gelatin.

[0152] Sterile injectable solutions can be prepared by incorporating the
active compound in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle, which contains
a basic dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable solutions, typical methods of preparation include
vacuum drying and freeze drying, which can yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.

[0153] Oral compositions generally include an inert diluent or an edible
carrier. For the purpose of oral therapeutic administration, the active
compound can be incorporated with excipients and used in the form of
tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions
can also be prepared using a fluid carrier for use as a mouthwash.
Pharmaceutically compatible binding agents, and/or adjuvant materials can
be included as part of the composition. The tablets, pills, capsules,
troches and the like can contain any of the following ingredients, or
compounds of a similar nature: a binder such as microcrystalline
cellulose, gum tragacanth or gelatin; an excipient such as starch or
lactose, a disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a glidant
such as colloidal silicon dioxide; a sweetening agent such as sucrose or
saccharin; or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring.

[0154] For administration by inhalation, the compounds can be delivered in
the form of an aerosol spray from a pressurized container or dispenser
which contains a suitable propellant, e.g., a gas such as carbon dioxide,
or a nebulizer.

[0155] Systemic administration of a therapeutic compound as described
herein can also be by transmucosal or transdermal means. For transmucosal
or transdermal administration, penetrants appropriate to the barrier to
be permeated are used in the formulation. Such penetrants are generally
known in the art, and include, for example, for transmucosal
administration, detergents, bile salts, and fusidic acid derivatives.
Transmucosal administration can be accomplished through the use of nasal
sprays. For transdermal administration, the active compounds are
formulated into ointments, salves, gels, or creams as generally known in
the art. In one embodiment, transdermal administration may be performed
my iontophoresis.

[0156] A therapeutic protein or peptide or a pharmaceutically acceptable
salt thereof, such as acetate salt or trifluoroacetate salt can be
formulated in a carrier system. The carrier can be a colloidal system.
The colloidal system can be a liposome, a phospholipid bilayer vehicle.
In one embodiment, the therapeutic peptide is encapsulated in a liposome
while maintaining peptide integrity. As one skilled in the art would
appreciate, there are a variety of methods to prepare liposomes. (See
Lichtenberg et al., Methods Biochem. Anal., 33:337-462 (1988); Anselem et
al., Liposome Technology, CRC Press (1993)). Liposomal formulations can
delay clearance and increase cellular uptake (See Reddy, Ann.
Pharmacother., 34(7-8):915-923 (2000)). An active agent can also be
loaded into a particle prepared from pharmaceutically acceptable
ingredients including, but not limited to, soluble, insoluble, permeable,
impermeable, biodegradable or gastroretentive polymers or liposomes. Such
particles include, but are not limited to, nanoparticles, biodegradable
nanoparticles, microparticles, biodegradable microparticles, nanospheres,
biodegradable nanospheres, microspheres, biodegradable microspheres,
capsules, emulsions, liposomes, micelles and viral vector systems.

[0157] The carrier can also be a polymer, e.g., a biodegradable,
biocompatible polymer matrix. In one embodiment, the therapeutic peptide
can be embedded in the polymer matrix, while maintaining protein
integrity. The polymer may be natural, such as polypeptides, proteins or
polysaccharides, or synthetic, such as poly α-hydroxy acids.
Examples include carriers made of, e.g., collagen, fibronectin, elastin,
cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin,
and combinations thereof. In one embodiment, the polymer is poly-lactic
acid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matrices
can be prepared and isolated in a variety of forms and sizes, including
microspheres and nanospheres. Polymer formulations can lead to prolonged
duration of therapeutic effect. (See Reddy, Ann. Pharmacother.,
34(7-8):915-923 (2000)). A polymer formulation for human growth hormone
(hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical
Biology, 2:548-552 (1998)).

[0159] In some embodiments, the therapeutic compounds are prepared with
carriers that will protect the therapeutic compounds against rapid
elimination from the body, such as a controlled release formulation,
including implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Such formulations can be prepared using known
techniques. The materials can also be obtained commercially, e.g., from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions
(including liposomes targeted to specific cells with monoclonal
antibodies to cell-specific antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared according to
methods known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811.

[0160] The therapeutic compounds can also be formulated to enhance
intracellular delivery. For example, liposomal delivery systems are known
in the art, see, e.g., Chonn and Cullis, "Recent Advances in Liposome
Drug Delivery Systems," Current Opinion in Biotechnology 6:698-708
(1995); Weiner, "Liposomes for Protein Delivery: Selecting Manufacture
and Development Processes," Immunomethods, 4(3):201-9 (1994); and
Gregoriadis, "Engineering Liposomes for Drug Delivery: Progress and
Problems," Trends Biotechnol., 13(12):527-37 (1995). Mizguchi et al.,
Cancer Lett., 100:63-69 (1996), describes the use of fusogenic liposomes
to deliver a protein to cells both in vivo and in vitro.

[0161] Dosage, toxicity and therapeutic efficacy of the therapeutic agents
can be determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose lethal
to 50% of the population) and the ED50 (the dose therapeutically
effective in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be expressed as
the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are
preferred. While compounds that exhibit toxic side effects may be used,
care should be taken to design a delivery system that targets such
compounds to the site of affected tissue in order to minimize potential
damage to uninfected cells and, thereby, reduce side effects.

[0162] The data obtained from the cell culture assays and animal studies
can be used in formulating a range of dosage for use in humans. The
dosage of such compounds lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity. The
dosage may vary within this range depending upon the dosage form employed
and the route of administration utilized. For any compound used in the
methods, the therapeutically effective dose can be estimated initially
from cell culture assays. A dose can be formulated in animal models to
achieve a circulating plasma concentration range that includes the IC50
(i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses in
humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.

[0163] Typically, an effective amount of the aromatic-cationic peptides or
a pharmaceutically acceptable salt thereof, such as acetate salt or
trifluoroacetate salt, e.g., SS-31 or a pharmaceutically acceptable salt
thereof, such as acetate salt or trifluoroacetate salt, sufficient for
achieving a therapeutic or prophylactic effect, range from about 0.000001
mg per kilogram body weight per day to about 10,000 mg per kilogram body
weight per day. Suitably, the dosage ranges are from about 0.0001 mg per
kilogram body weight per day to about 100 mg per kilogram body weight per
day. For example dosages can be 1 mg/kg body weight or 10 mg/kg body
weight every day, every two days, or every three days or within the range
of 1-10 mg/kg every week, every two weeks or every three weeks. In one
embodiment, a single dosage of peptide ranges from 0.001-10,000
micrograms per kg body weight. In one embodiment, aromatic-cationic
peptide concentrations in a carrier range from 0.2 to 2000 micrograms per
delivered milliliter. An exemplary treatment regime entails
administration once per day or once a week. In therapeutic applications,
a relatively high dosage at relatively short intervals is sometimes
required until progression of the disease is reduced or terminated, and
preferably until the subject shows partial or complete amelioration of
symptoms of disease. Thereafter, the patient can be administered a
prophylactic regime.

[0164] By way of example, and not by way of limitation, in one embodiment
for the prevention or amelioration of MV-induced diaphragm weakness, an
initial dose of cationic peptide (e.g., SS-31 or a pharmaceutically
acceptable salt thereof, such as acetate salt or trifluoroacetate salt)
is administered at about 1-20 mg/kg, about 1-15 mg/kg, about 1-10 mg/kg,
about 1-5 mg/kg, 2-15 mg/kg, about 2-10 mg/k, about 2-5 mg/kg, about 2-3
mg/kg, or about 3 mg/kg. The initial dose is administered prior to, or
shortly after MV begins. Additionally or alternatively, the initial dose
is followed by a dose of about 0.01 mg/kg per hour, about 0.02 mg/kg per
hour, about 0.03 mg/kg per hour, about 0.04 mg/kg per hour, about 0.05
mg/kg per hour, about 0.06 mg/kg per hour, about 0.07 mg/kg per hour,
about 0.08 mg/kg per hour, about 0.09 mg/kg per hour, about 0.1 mg/kg per
hour, about 0.2 mg/kg per hour, about 0.3 mg/kg per hour, about 0.5 mg/kg
per hour, about 0.75 mg/kg per hour or about 1.0 mg/kg per hour.

[0165] In some embodiments, a therapeutically effective amount of an
aromatic-cationic peptide or a pharmaceutically acceptable salt thereof,
such as acetate salt or trifluoroacetate salt may be defined as a
concentration of peptide at the target tissue of 10-12 to 10-6
molar, e.g., approximately 10-7 molar. This concentration may be
delivered by systemic doses of 0.001 to 100 mg/kg or equivalent dose by
body surface area. The schedule of doses would be optimized to maintain
the therapeutic concentration at the target tissue, most preferably by
single daily or weekly administration, but also including continuous
administration (e.g., parenteral infusion or transdermal application).

[0166] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a subject,
including but not limited to, the severity of the disease or disorder,
previous treatments, the general health and/or age of the subject, and
other diseases present. Moreover, treatment of a subject with a
therapeutically effective amount of the therapeutic compositions
described herein can include a single treatment or a series of
treatments.

[0167] The mammal treated in accordance present methods can be any mammal,
including, for example, farm animals, such as sheep, pigs, cows, and
horses; pet animals, such as dogs and cats; laboratory animals, such as
rats, mice and rabbits. In one embodiment, the mammal is a human.

[0168] In one embodiment, an additional therapeutic agent is administered
to a subject in combination with an aromatic cationic peptide or a
pharmaceutically acceptable salt thereof, such as acetate salt or
trifluoroacetate salt, such that a synergistic therapeutic effect is
produced. A "synergistic therapeutic effect" refers to a
greater-than-additive therapeutic effect which is produced by a
combination of two therapeutic agents, and which exceeds that which would
otherwise result from individual administration of either therapeutic
agent alone. Therefore, lower doses of one or both of the therapeutic
agents may be used in treating muscle infirmities, resulting in increased
therapeutic efficacy and decreased side-effects.

[0169] The multiple therapeutic agents may be administered in any order,
simultaneously, sequentially or overlapping. If simultaneously, the
multiple therapeutic agents may be provided in a single, unified form, or
in multiple forms (by way of example only, either as a single pill or as
two separate pills). One of the therapeutic agents may be given in
multiple doses, or both may be given as multiple doses. If not
simultaneous, the timing between the multiple doses may vary from more
than zero weeks to less than four weeks. In addition, the combination
methods, compositions and formulations are not to be limited to the use
of only two agents.

EXAMPLES

[0170] The present invention is further illustrated by the following
examples, which should not be construed as limiting in any way.

I. Example 1

[0171] A. Experimental Design

[0172] The purpose of this experiment was to demonstrate the role that
mitochondrial ROS emission plays in MV-induced diaphragmatic weakness,
and to demonstrate the effect of a mitochondrial-targeted antioxidant
peptide (SS-31) on mitochondrial function and diaphragm muscle in rats.
Two different groups of rats (1 and 2) were treated as follows.

[0173] 1. Awake and Spontaneously Breathing Rats

[0174] To determine the effect of a mitochondrial-targeted antioxidant
(SS-31) on diaphragmatic contractile function, fiber cross sectional area
(CSA), and mitochondrial function in awake and spontaneously breathing
rats, animals were treated as follows. Animals (n=6/group) were randomly
assigned into one of two experimental groups: (1) Control group-injected
with saline (i.p.) at three hour intervals for 12 hours; and (2)
Mitochondrial antioxidant group-injected (i.p.) with SS-31 every three
hours for 12 hours. At the completion of the 12-hour treatment periods,
diaphragmatic contractile function, fiber CSA, mitochondrial ROS
emission, and mitochondrial respiratory function were measured.

[0175] The mitochondrial-targeted antioxidant SS-31 was dissolved in
saline and delivered via four subcutaneous injections during the 12-hour
experimental period. The first bolus (loading) dose (3 mg/kg;
subcutaneous injection) was administered at the onset of the experiment.
SS-31 (0.05 mg/kg/hr) was then administered via subcutaneous injections
staged every three hours during the 12-hour experiment. All animals
received the same total amount of SS-31 during 12 hours for all
experiments requiring SS-31 administration.

[0176] 2. Anesthetized Rats

[0177] To analyze mitochondrial ROS emissions following MV-induced
diaphragmatic oxidative stress and weakness, rats were randomly assigned
to one of three experimental groups (n=12/group): (1) an acutely
anesthetized control group; (2) a 12-hour MV group (MV); and 3) a 12-hour
MV group treated with the mitochondrial-targeted antioxidant SS-31
(MVSS). Because of the large tissue requirement for our numerous
dependent measures, six animals from each experimental group were used
for the mitochondrial measures and the remaining six animals in each
group were employed in all other biochemical assays.

[0178] Animals in the control group were acutely anesthetized with an
intraperitoneal (IP) injection of sodium pentobarbital (60 mg/kg body
weight). After reaching a surgical plane of anesthesia, the diaphragms
were quickly removed. In one group of animals (n=6), a strip of the
medial costal diaphragm was immediately used for in vitro contractile
measurements, a separate section was stored for histological
measurements, and the remaining portions of the costal diaphragm were
rapidly frozen in liquid nitrogen and stored at -80° C. for
subsequent biochemical analyses. In a second group of animals (n=6), the
entire costal diaphragm was rapidly removed and used to isolate
mitochondria for measurements of mitochondrial respiration and ROS
emission. The mitochondrial-targeted antioxidant SS-31 was dissolved in
saline and delivered in a bolus (loading) dose (3 mg/kg; subcutaneous
injection) 15 min prior to initiation of MV. A constant intravenous
infusion (0.05 mg/kg/hr) of SS-31 was maintained throughout MV.

[0181] A mitochondria-targeted antioxidant designated as "SS-31" was
selected for use in the current experiments. This molecule belongs to a
family of small, water soluble peptides that contain an alternating
aromatic-cationic motif and selectively target to the mitochondria. See,
e.g., Zhao et al., Cell-permeable peptide antioxidants targeted to inner
mitochondrial membrane inhibit mitochondrial swelling, oxidative cell
death, and reperfusion injury. The Journal of biological chemistry. Vol.,
279(33): 34682-34690 (2004).

[0184] The carotid artery was cannulated to permit the continuous
measurement of blood pressure and the collection of blood during the
protocol. Arterial blood samples (100 μl per sample) were removed
periodically and analyzed for arterial pO2, pCO2 and pH using
an electronic blood-gas analyzer (GEM Premier 3000; Instrumentation
Laboratory, Lexington, Mass.). Ventilator adjustments were made if
arterial PCO2 exceeded 40 mm Hg. Arterial PO2 was
maintained>60 mmHg throughout the experiment by increasing the
FIO2 (22-26% oxygen).

[0185] A venous catheter was inserted into the jugular vein for continuous
infusion of sodium pentobarbital (˜10 mg/kg/hr) and fluid
replacement. Body temperature was maintained at 37° C. by use of a
recirculating heating blanket and heart rate was monitored via a lead II
electrocardiograph. Continuous care during the MV protocol included
lubricating the eyes, expressing the bladder, removing airway mucus,
rotating the animal, and passively moving the limbs. Animals also
received an intramuscular injection of glycopyrrolate (0.18 mg/kg) every
two hours during MV to reduce airway secretions. Upon completion of MV,
in one group of six animals the diaphragm was quickly removed and a strip
of the medial costal diaphragm was used for in vitro contractile
measurements, a section was stored for histochemical analyses, and the
remaining portion was frozen in liquid nitrogen and stored at -80°
C. for subsequent analyses. In an additional group of animals (n=6), the
entire costal diaphragm was rapidly removed and used to isolate
mitochondria for measurements of mitochondrial respiration and ROS
emission.

[0195] The levels of reactive carbonyl derivatives in the myofibrillar
protein samples were assessed as an index of the magnitude of protein
modification. This was accomplished using the Oxyblot Oxidized Protein
Detection Kit from Chemicon International (Temecula, Ca) as described
previously. See Kavazis et al. (2009).

[0196] RNA Isolation and cDNA Synthesis.

[0197] Total RNA was isolated from muscle tissue with TRIzol Reagent (Life
Technologies, Carlsbad, Calif.) according to the manufacturer's
instructions. RNA content (μg/mg muscle) was evaluated by
spectrophotometry. RNA (5 μg) was then reverse transcribed with the
Superscript III First-Strand Synthesis System for RT-PCR (Life
Technologies), using oligo(dT)20 primers and the protocol outlined by the
manufacturer.

[0201] A section of the ventral costal diaphragm was homogenized and the
in vitro chymotrypsin-like activity of the 20S proteasome was measured
fluorometrically using techniques described by Stein and co-workers. See
Stein et al., Kinetic characterization of the chymotryptic activity of
the 20S proteasome. Biochemistry 35(13): 3899-3908 (1996).

[0202] Functional Measures.

[0203] Measurement of in vitro diaphragmatic contractile properties. At
the completion of the experimental periods, the entire diaphragm was
removed and placed in a dissecting chamber containing a Krebs-Hensleit
solution equilibrated with 95% O2-5% CO2 gas. A muscle strip
(˜3 mm wide), including the tendinous attachments at the central
tendon and rib cage was dissected from the midcostal region. The strip
was suspended vertically between two lightweight Plexiglas clamps with
one end connected to an isometric force transducer (model FT-03, Grass
Instruments, Quincy, Mass.) within a jacketed tissue bath. The muscle was
electrically stimulated to contract and the force output was recorded via
a computerized data-acquisition system as previously described. See
Powers et al., Mechanical ventilation results in progressive contractile
dysfunction in the diaphragm. J Appl Physiol, Vol. 92(5):1851-1858
(2002). For comparative purposes, diaphragmatic (bundles of fibers) force
production was normalized as fiber cross sectional area (i.e., specific
force production).

[0207] Comparisons between groups for each dependent variable were made by
a one-way analysis of variance (ANOVA) and, when appropriate, a Tukey HSD
(honestly significant difference) test was performed post-hoc.
Significance was established at p<0.05. Data are presented as
means±SEM.

[0208] Measurement of Mitochondrial Protein Carbonyl Groups.

[0209] For mitochondrial protein extraction, ventricular tissues were
homogenized in mitochondrial isolation buffer (1 mM EGTA, 10 mM HEPES,
250 mM sucrose, 10 mM Tris-HCl, pH 7.4). The lysates were centrifuged for
7 min at 800 g in 4° C. The supernatants were then centrifuged for
30 min at 4000 g in 4° C. The crude mitochondria pellets were
resuspended in small volume of mitochondrial isolation buffer, sonicated
on ice to disrupt the membrane, and treated with 1% streptomycin sulfate
to precipitate mitochondrial nucleic acids. The OxiSelect® Protein
Carbonyl ELISA Kit (Cell Biolabs) was used to analyze 1 μg of protein
sample per assay. The ELISA was performed according to the instruction
manual, with slight modification. Briefly, protein samples were reacted
with dinitrophenylhydrazine (DNPH) and probed with anti-DNPH antibody,
followed by HRP conjugated secondary antibody. The anti-DNPH antibody and
HRP conjugated secondary antibody concentrations were 1:2500 and 1:4000,
respectively.

[0213] The NADPH oxidase assay was performed as follows. In brief, 10
μg of ventricular protein extract was incubated with dihydroethidium
(DHE, 10 μM), sperm DNA (1.25 μg/ml), and NADPH (50 μM) in
PBS/DTPA (containing 100 μM DTPA), The assay was incubated at
37° C. in the dark for 30 min and the fluorescence was detected
using excitation/emission of 490/580 nm.

[0216] To determine the impact of the mitochondrial antioxidant SS-31 on
diaphragmatic contractile function, fiber cross sectional area (CSA), and
mitochondrial function in awake and spontaneously breathing rats, animals
were treated for 12-hours with the same levels of SS-31 that were
provided to the mechanically ventilated animals during the 12-hour MV
period. The results shown below in Tables 7A-7C demonstrate that,
compared to untreated control animals, the treatment of animals with
SS-31 does not influence diaphragmatic mitochondrial ROS emission and the
mitochondrial respiratory ratio. Further, the results demonstrate that
compared to control, treatment of animals with SS-31 did not alter
diaphragmatic contractile function and fiber CSA.

[0217] Table 7A shows fiber cross-sectional area (CSA) in diaphragm muscle
fibers from both control (treated with saline injections) and awake and
spontaneously breathing animals treated with the mitochondrial-targeted
antioxidant SS-31. No significant differences in diaphragmatic fiber CSA
existed between the Control and SS-31 groups in any fiber type. Values
are means±SEM.

[0218] Table 7B shows the effects of a mitochondrial targeted antioxidant
(SS-31) on the diaphragmatic force-frequency response (in vitro) in
control (saline injected) and SS-31 treated animals. No significant
differences in diaphragmatic force production existed between the control
and SS-31 groups at any stimulation frequency. Values are means±SEM.

[0219] Table 7C shows the effects of a mitochondrial targeted antioxidant
(SS-31) on diaphragm mitochondrial hydrogen peroxide emission and the
mitochondrial respiratory function in control (saline injected) and SS-31
treated animals. These data were obtained using pyruvate/malate as
substrate. VO2=mitochondrial oxygen consumption; RCR=respiratory
control ratio. Units for state-3 and state-4 VO2 are nmoles
oxygen/mg protein/minute. Values are means±SEM *=different from
control at p<0.05.

[0220] 2. Physiological Responses to Prolonged MV

[0221] To assess the efficacy of the MV protocol for maintaining
homeostasis, arterial blood pressures, arterial PCO2, arterial
PO2 and arterial pH were measured in all animals at the beginning of
the experiments and at various time intervals during MV. Although small
variations in arterial blood pressure, blood gases, and pH existed over
time, our results confirm that arterial blood pressure and blood-gas/pH
homeostasis were well-maintained during MV (Table 8).

[0222] Table 8 shows animal heart rates, systolic blood pressure, and
arterial blood gas tension/pH and at the completion of 12 hours of
mechanical ventilation. Values are means±SEM. No significant
differences existed between the two experimental groups in any of these
physiological variables.

[0223] In addition, strict aseptic techniques were followed throughout the
experiments given that sepsis is associated with diaphragmatic
contractile dysfunction. Importantly, the data illustrate that animals
did not develop infection during MV. This is supported by the observation
that microscopic examination of blood revealed no detectable bacteria,
and that postmortem (visual) examination of the lungs and peritoneal
cavity yielded no detectable abnormalities. Furthermore, MV animals were
afebrile during the investigation, with body temperatures ranging from
36.3 to 37.4° C. Finally, during the course of MV, no significant
(P<0.05) changes occurred in the body weights of the MV animals.
Collectively, these results indicate that the MV animals were
significantly free of any infection.

[0224] As compared to controls, the results show that treatment of
spontaneous breathing animals with SS-31 did not alter any of these
dependent measures (see below). Therefore, further experiments were
performed using SS-31 as a mitochondrial-targeted antioxidant to analyze
mitochondrial ROS emissions during MV-induced diaphragmatic weakness,
which consisted of MV for 12-hours.

[0226] Mitochondrial-derived ROS emissions were assessed in mitochondria
for an association with MV-induced oxidative damage, contractile
dysfunction, and atrophy in the diaphragm. In this respect, rats were
treated with a mitochondrial-targeted antioxidant (SS-31) to prevent
MV-induced ROS emission from diaphragm mitochondria. It is noted that
treatment with SS-31 prevented the MV-induced increase in diaphragmatic
mitochondrial H2O2 release both during state 3 and 4
mitochondrial respiration. See FIG. 1. In this regard, hydrogen peroxide
release from mitochondria isolated from diaphragms of mechanically
ventilated (MV) rats, in the absence of SS-31 did not show a decrease. As
such, treatment of animals with SS-31 significantly reduced the rates of
H2O2 release from the mitochondria following prolonged MV.
Values are mean±SEM. *=different (p<0.05) from both CON and MVSS
(n=6/group). See FIG. 1.

[0227] Prolonged MV results in damage to mitochondria as indicated by
impaired coupling (i.e., lower respiratory control ratios) in
mitochondria isolated from the diaphragm of MV animals. Therefore,
treatment of animals with SS-31 protects diaphragmatic mitochondria from
MV-induced mitochondrial uncoupling. As shown in Table 9, treatment with
SS-31 was successful in averting diaphragmatic mitochondrial uncoupling
that occurs following prolonged MV.

[0230] To determine if mitochondrial ROS emission is required for
MV-induced oxidative stress in the diaphragm, two biomarkers of oxidative
damage were measured, i.e., diaphragmatic levels of 4-HNE-conjugated
cytosolic proteins and levels of protein carbonyls in myofibrillar
proteins. The results reveal that treatment of animals with SS-31
protected the diaphragm against the ROS-induced increase in both protein
carbonyls and 4-HNE-conjugated proteins normally associated with
prolonged MV. See FIG. 2. In this respect, levels of oxidatively modified
proteins in the diaphragm of control (CON), mechanically ventilated (MV),
and mechanically ventilated rats treated with the mitochondrial-targeted
antioxidant SS-31 (MVSS) were measured.

[0231] As shown in FIG. 2A, levels of 4-hydroxyl-nonenal-conjugated
proteins in the diaphragm of the three experimental groups are listed.
The image above the histograph is a representative western blot of data
from the three experimental groups. FIG. 2B further illustrates the
levels of protein carbonyls in the diaphragm of the three experimental
groups. *=different (p<0.05) from both CON and MVSS (n=6/group). See
FIG. 2.

[0233] To assess the role that mitochondrial ROS emission plays in
MV-induced diaphragmatic contractile dysfunction, diaphragmatic
contractile performance in vitro using strips of diaphragm muscle
obtained from control, MV, and MV animals treated with SS-31 were
measured. Prevention of mitochondrial ROS emission using SS-31
successfully prevented the diaphragmatic contractile dysfunction
associated with prolonged MV. See FIG. 3. As shown in FIG. 3, prolonged
MV effects the diaphragmatic force-frequency response (in vitro) in
control and mechanically ventilated rats with/without mitochondrial
targeted antioxidants. However, no significant differences in
diaphragmatic force production existed between the CON and MVSS groups at
any stimulation frequency. Values are means±SEM. Note that some of the
SEM bars are not visible because of the small size. *=different
(p<0.05) from both CON and MVSS (n=6/group). See FIG. 3.

[0234] MV-induced oxidative stress is a requirement for the diaphragmatic
fiber atrophy that is associated with prolonged MV. See Betters et al.,
Trolox attenuates mechanical ventilation-induced diaphragmatic
dysfunction and proteolysis. Am J Respir Crit Care Med., Vol.,
170(11):1179-1184 (2004). As shown in FIG. 4, fiber cross-sectional area
(CSA) in diaphragm muscle myofibers from control (CON) and mechanically
ventilated rats with (MVSS) and without mitochondrial targeted
antioxidants (MV) were tested. It is noted that no significant
differences in diaphragmatic fiber CSA existed between the CON and MVSS
groups in any fiber type. Values are means±SEM. *=different
(p<0.05) from both CON and MVSS (n=6/group). See FIG. 4. It was
determined that MV-induced mitochondrial ROS emission is a requirement
for MV-induced diaphragmatic atrophy. Myofiber cross-sectional area was
determined for individual fiber types for all treatment groups. The data
indicates that prevention of the MV-induced increase in mitochondrial ROS
emission protects the diaphragm from MV-induced fiber atrophy. See FIG.
4.

[0236] The ubiquitin-proteasome system of proteolysis is activated in the
diaphragm during prolonged MV and therefore likely contributes to
MV-induced diaphragmatic protein breakdown. To determine the effects of
mitochondrial ROS emission on the ubiquitin-proteasome system of
proteolysis, 20S proteasome activity was measured along with both mRNA
and protein levels of two important muscle specific E3 ligases (i.e.,
atrogin-1/MAFbx and MuRF-1) in the diaphragm. The results reveal that
prevention of MV-induced mitochondrial ROS release via SS-31 prevented
the MV-induced increase in 20S proteasome activity in the diaphragm. See
FIG. 5A. Further, the results indicate that prolonged MV resulted in a
significant increase in atrogin-1/MAFbx mRNA levels in the diaphragm of
both MV groups; however, treatment of animals with SS-31 significantly
blunted the MV-induced increase in atrogin-1/MAFbx protein levels in the
diaphragm. See FIG. 5B.

[0237] FIG. 5C illustrates the impact of prolonged MV on both
diaphragmatic mRNA and protein levels of MuRF-1. Prolonged MV resulted in
a significant increase in MuRF-1 mRNA levels in the diaphragm and
although MuRF-1 proteins levels tended to increase in the diaphragm of
mechanically ventilated animals, these differences did not reach
significance. The images above the histograms in FIG. 5B-C are
representative western blots of data from the three experimental groups.
Values are means±SEM. *=different (p<0.05) from both CON and MVSS.
**=different (p<0.05) from both CON and MV (n=6/group). See FIG. 5.

[0238] Calpain and caspase-3 activation in the diaphragm has an important
role in MV-induced diaphragmatic atrophy and contractile dysfunction. See
McClung et al., Caspase-3 regulation of diaphragm myonuclear domain
during mechanical ventilation-induced atrophy, Am J Respir Crit Care
Med., Vol. 175(2):150-159 (2007). Diaphramatic calpain and caspase-3
activity were assayed using two different but complimentary methods.
First, active calpain-1 and caspase-3 levels in the muscle were
determined via Western blot to detect the cleaved and active forms of
calpain 1 and caspase-3. See FIG. 6. As shown in FIG. 6A, the active form
of calpain 1 in diaphragm muscle is detected at the completion of 12
hours of MV. The cleaved and active band of caspase-3 in diaphragm muscle
at the completion of 12 hours of MV is also illustrated. See FIG. 6B. The
images above the histograms in FIGS. 6A and 6B are representative western
blots of data from the three experimental groups. Values are
means±SEM. *=different (p<0.05) from both CON and MVSS (n=6/group).
See FIG. 6B.

[0239] Calpain 1 and caspase-3 activity were measured at one time period.
Therefore, calpain and caspase-3 specific degradation products of
αII-spectrin were also measured as these breakdown products provide
an in vivo signature that can be detected. See FIG. 7. This technique
provides an index of in vivo calpain and caspase-3 activity in the
diaphragm over a prolonged period of time during MV. As shown in FIG. 7A,
levels of the 145 kDa α-II-spectrin degradation product (SBPD) in
diaphragm muscle following 12 hours of MV are measured. It is noted that
the SBDP 145 kDa is an α-II-spectrin degradation product specific
to calpain cleavage of intact α-II-spectrin and therefore, the
cellular level of SBDP 145 kDa is employed as a biomarker of in vivo
calpain activity.

[0240] As shown in FIG. 7B, levels of the 120 kDa α-II-spectrin
break-down product (SBPD 120 kDa) in diaphragm muscle following 12 hours
of MV were measured. It is noteworthy that the SBDP 120 kDa is a
α-II-spectrin degradation product specific to caspase-3 cleavage of
intact α-II-spectrin and therefore, the cellular levels of SBDP 120
kDa can be used as a biomarker of caspase-3 activity. The images above
the FIGS. 7A and 7B histograms are representative western blots of data
from the three experimental groups. Values are means±SEM. *=different
(p<0.05) from both CON and MVSS (n=6/group). See FIG. 7.

[0241] Together these results demonstrate that treatment of animals with a
mitochondrial-targeted antioxidant (SS-31) protected the diaphragm
against the activation of both calpain and caspase-3. See FIGS. 6-7.
These findings illustrate that mitochondria are the dominant source of
MV-induced ROS production in the diaphragm and that mitochondrial ROS
production is essential for MV-induced activation of both calpain and
caspase-3 in the diaphragm.

[0243] After demonstrating that prevention of MV-induced increases in
mitochondrial ROS emission protects the diaphragm against protease
activation, the relative abundance of the sarcomeric protein actin in the
diaphragm as a marker of disuse-induced muscle proteolysis was measured.
Since actin is preferentially degraded during disuse muscle atrophy,
assessment of the actin protein levels provides an index of proteolysis.
See Li et al., Interleukin-1 stimulates catabolism in C2Cl2 myotubes.
American Journal of Physiology., Vol., 297(3):C706-714 (2009). The
results reveal that, compared to diaphragm muscle from both control and
MV-SS animals, the actin abundance was significantly reduced in diaphragm
muscle from animals exposed to prolonged MV without mitochondrial
antioxidants. See FIG. 8. Therefore, prevention of MV-induced
mitochondrial ROS emission not only protected against protease
activation, this treatment protected against MV-induced diaphragmatic
proteolysis.

[0244] As shown in FIG. 8, the ratio of actin to total sarcomeric protein
levels in the diaphragm from control (CON) and mechanically ventilated
animals with (MVSS) without mitochondrial-targeted antioxidants (MV) was
measured. Because actin is preferentially degraded during disuse muscle
atrophy, assessment of the ratio of actin to total sarcomeric protein
levels provides a relative index of diaphragmatic proteolysis during
prolonged MV. The image above the histogram is a representative western
blot of data from the three experimental groups. Values are means±SEM.
*=different (p<0.05) from both CON and MVSS (n=6/group). See FIG. 8.

II. Example 2

[0245] A. Experimental Design and Methods:

[0246] The purpose of this example was to demonstrate that MV-induced
mitochondrial oxidation is generalizable to disuse-induced skeletal
muscle weakness. Two different groups of mice (1 and 2) were treated as
follows.

[0247] 1. Normal, Mobile Mice

[0248] Normal, mobile mice were randomly divided into two groups, A and B,
with 8 mice per group. Group A mice received an an injection of sterile
saline; Group B mice received an injection of the mitochondrial targeted
peptide SS-31.

[0249] 2. Hindlimb Casted Mice

[0250] Mouse hind limbs were immobilized by casting for 14 days, thereby
inducing hind limb muscle atrophy. Casted mice received an injection of
sterile saline (0.3 ml) or an injection containing the peptide SS-31 (0.3
ml). A control group of untreated mice was also used in this experiment.

[0251] B. Materials and Methods:

Animals

[0252] Seventy-two adult male C57B16 mice (age 21-28 weeks, body weight
26.44±0.54 g) were used in these experiments. Animals were maintained
on a 12:12 hour light-dark cycle and provided food (AIN93 diet) and water
ad libitum throughout the experimental period. The Institutional Animal
Care and Use Committee of the University of Florida approved these
experiments.

Experimental Design

[0253] To test the hypothesis that mitochondrial ROS production plays a
role in immobilization-induced skeletal muscle atrophy, mice were
randomly assigned to one of three experimental groups (n=24/group): 1) no
treatment (Control) group; 2) 14 days of hind-limb immobilization group
(Cast); and 3) 14 days of hind-limb immobilization group treated with the
mitochondrial-targeted antioxidant SS-31 (Cast+SS). Note that 14-days of
hind-limb immobilization group (Cast) received saline infusions whereas
animals in the group were treated with the mitochondrial-targeted
antioxidant SS-31 during immobilization period.

Experimental Protocol

[0254] Immobilization.

[0255] Mice were anesthetized with gaseous isoflurane (3% induction,
0.5-2.5% maintenance). Anesthetized animals were cast-immobilized
bilaterally with the ankle joint in the plantar-flexed position to induce
maximal atrophy of the soleus and plantaris muscle. Both hindlimbs and
the caudal fourth of the body were encompassed by a plaster of paris
cast. A thin layer of padding was placed underneath the cast in order to
prevent abrasions. In addition, to prevent the animals from chewing on
the cast, one strip of fiberglass material was applied over the plaster.
The mice were monitored on a daily basis for chewed plaster, abrasions,
venous occlusion, and problems with ambulation.

[0261] Respiration was measured polarographically in a respiration chamber
maintained at 37° C. (Hansatech Instruments, United Kingdom).
After the respiration chamber was calibrated, permeabilized fiber bundles
were incubated with 1 ml of respiration buffer Z containing 20 mM
creatine to saturate creatine kinase (Saks V A, et al. Permeabilized cell
and skinned fiber techniques in studies of mitochondrial function in
vivo. Mol Cell Biochem 184: 81-100, 1998; Walsh B, et al. The role of
phosphorylcreatine and creatine in the regulation of mitochondrial
respiration in human skeletal muscle. J Physiol 537: 971-978, 2001). Flux
through complex I was measured using 5 mM pyruvate and 2 mM malate. The
maximal respiration (state 3), defined as the rate of respiration in the
presence of ADP, was initiated by adding 0.25 mM ADP to the respiration
chamber. Basal respiration (state 4) was determined in the presence of 10
μg/ml oligomycin to inhibit ATP synthesis. The respiratory control
ratio (RCR) was calculated by dividing state 3 by state 4 respiration.

[0262] Mitochondrial ROS Production.

[0263] Mitochondrial ROS production was determined using Amplex® Red
(Molecular Probes, Eugene, Oreg.). The assay was performed at 37°
C. in 96-well plates using succinate as the substrate. Specifically, this
assay was developed on the concept that horseradish peroxidase catalyzes
the H2O2-dependent oxidation of non-fluorescent Amplex® Red
to fluorescent Resorufin Red, and it is used to measure H2O2 as
an indicator of superoxide production. Superoxide dismutase (SOD) was
added at 40 units/ml to convert all superoxide into H2O2. We
monitored Resorufin formation (Amplex® Red oxidation by
H2O2) at an excitation wavelength of 545 nm and an emission
wavelength of 590 nm using a multiwell plate reader fluorometer
(SpectraMax, Molecular Devices, Sunnyvale, Calif.). The level of
Resorufin formation was recorded every 5 minutes for 15 minutes, and
H2O2 production was calculated with a standard curve.

[0264] Western Blot Analysis.

[0265] Protein abundance was determined in skeletal samples via Western
Blot analysis. Briefly, soleus and plataris tissue samples were
homogenized 1:10 (wt/vol) in 5 mM Tris (pH 7.5) and 5 mM EDTA (pH 8.0)
with a protease inhibitor cocktail (Sigma) and centrifuged at 1500 g for
10 min at 4° C. After collection of the resulting supernatant,
muscle protein content was assessed by the method of Bradford (Sigma, St.
Louis). Proteins were separated using electrophoresis via 4-20%
polyacrylamide gels containing 0.1% sodium dodecyl sulfate for ˜1 h
at 200 V. After electrophoresis, the proteins were transferred to
nitrocellulose membranes and incubated with primary antibodies directed
against the protein of interest. 4-HNE (Abcam) was probed as a
measurement indicative of oxidative stress while proteolytic activity was
assessed by cleaved (active) calpain-1 (Cell Signaling) and cleaved
(active) caspase-3 (Cell Signaling). Following incubation, membranes were
washed with PBS-Tween and treated with secondary antibody (Amersham
Biosciences). A chemiluminescent system was used to detect labeled
proteins (GE Healthcare) and membranes were developed using
autoradiography film and a developer (Kodak). The resulting images were
analyzed using computerized image analysis to determine percentage change
from control. Membranes were stained with Ponceau S and analyzed to
verify equal protein loading and transfer.

[0268] Comparisons between groups for each dependent variable were made by
a one-way analysis of variance (ANOVA) and, when appropriate, a Tukey HSD
(honestly significant difference) test was performed post-hoc.
Significance was established at p<0.05. Data are presented as
means±SEM.

[0269] C. Results:

[0270] As shown in FIGS. 9-18, SS-31 had no effect on normal skeletal
muscle size or mitochondrial function. However, SS-31 was able to prevent
oxidative damage and associated muscle weakness (e.g., atrophy,
contractile dysfunction, etc.) emanating from hind limb immobilization.

[0271] 1. Normal, Mobile Mice

[0272] As illustrated by FIG. 9A-D, SS-31 had no effect on soleus muscle
weight, the respiratory coupling ratio (RCR), mitochondrial state 3
respiration, or mitochondrial state 4 respiration, respectively in mobile
mice. RCR is the respiratory quotient ratio of state 3 to state 4
respiration, as measured by oxygen consumption. Likewise, FIG. 10A-C show
that SS-31 did not have any variable effects on muscle fibers of
different size in normal soleus muscle. Furthermore, as illustrated by
FIG. 11A-D, SS-31 had no effect on plantaris muscle weight, the
respiratory coupling ratio (RCR), mitochondrial state 3 respiration, or
mitochondrial state 4 respiration, respectively. Similarly, FIG. 12A-B
shows that SS-31 did not impart any variable effects to the muscle fibers
of different size in normal plantaris muscle fiber tissue.

[0273] 2. Hindlimb Casted Mice

[0274] As shown by FIG. 13A-D, casting for 7 days led to a significant
decrease in soleus muscle weight (FIG. 13A), RCR (FIG. 13B), and
mitochondrial state 3 respiration (FIG. 13C), all of which was reversed
by administration of SS-31. The casting did not have a significant effect
on state 4 respiration. Likewise, casting for 7 days significantly
increased H2O2 production by mitochondria isolated from soleus
muscle, which was similarly prevented by SS-31. See FIG. 14A-B. As shown
in FIG. 14B, SS-31 prevented cross sectional area loss for three types of
fibers in the soleus (type I, IIa and IIb/x).

[0275] Casting also significantly increased oxidative damage in soleus
muscle, as measured by lipid peroxidation via 4-hydroxynonenal (4-HNE).
See FIG. 15A. This effect was overcome by SS-31 administration. Moreover,
casting significantly increased protease activity in the soleus muscle,
which likely accounts for the muscle degradation and atrophy. As shown in
FIG. 15B-D, calpain-1, caspase-3 and caspase-12 proteolytic degradation
of muscle, respectively, were all prevented by SS-31.

[0276] As illustrated by FIG. 16A-D, casting for 7 days leads to a
significant decrease in plantaris muscle weight (FIG. 16A), RCR (FIG.
16B), and mitochondrial state 4 respiration (FIG. 16D), which is closely
associated with ROS generation. All such effects were reversed via SS-31
administration. The casting did not have a significant effect on state 3
respiration. See FIG. 16C. Similarly, casting for 7 days significantly
increased H2O2 production by mitochondria isolated from
plantaris muscle, which was prevented by SS-31. See FIG. 17A-B. As shown
in FIG. 17B, SS-31 prevented cross sectional area loss for two types of
fibers in the plantaris (type Ha and IIb/x).

[0277] Casting also significantly increased oxidative damage in plantaris
muscle, as measured by lipid peroxidation via 4-hydroxynonenal (4-HNE).
See FIG. 18A. This effect was overcome by SS-31 administration. Moreover,
casting significantly increased protease activity in the soleus muscle,
which likely accounts for the muscle degradation and atrophy. As shown in
FIG. 18B-D, calpain-1, caspase-3 and caspase-12 proteolytic degradation
of muscle were all prevented by SS-31, respectively.

[0278] In summary, results from these examples show that administering
SS-31 to subjects with MV-induced or disuse-induced increases in
mitochondrial ROS emissions not only reduces protease activity, but also
attenuates skeletal muscle atrophy and contractile dysfunction. Treatment
of animals with the mitochondrial-targeted antioxidant SS-31 was
successful in preventing the atrophy in type I, IIa, and IIx/b fibers in
the skeletal muscles described above. Further, prevention of MV-induced
and disuses-induced increases in mitochondrial ROS emission also
protected the diaphragm against MV-induced decreases in diaphragmatic
specific force production at both sub-maximal and maximal stimulation
frequencies. See FIG. 3. Together, these results indicate that SS-31 can
protect against and treat MV-induced and disuse-induced mitochondrial ROS
emission in the diaphragm and other skeletal muscles.

[0279] The present invention is not to be limited in terms of the
particular embodiments described in this application, which are intended
as single illustrations of individual aspects of the invention. Many
modifications and variations of this invention can be made without
departing from its spirit and scope, as will be apparent to those skilled
in the art. Functionally equivalent methods and apparatuses within the
scope of the invention, in addition to those enumerated herein, will be
apparent to those skilled in the art from the foregoing descriptions.
Such modifications and variations are intended to fall within the scope
of the appended claims. The present invention is to be limited only by
the terms of the appended claims, along with the full scope of
equivalents to which such claims are entitled. It is to be understood
that this invention is not limited to particular methods, reagents,
compounds compositions or biological systems, which can, of course, vary.
It is also to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not intended to
be limiting.

[0280] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of any
individual member or subgroup of members of the Markush group.

[0281] As will be understood by one skilled in the art, for any and all
purposes, particularly in terms of providing a written description, all
ranges disclosed herein also encompass any and all possible subranges and
combinations of subranges thereof. Any listed range can be easily
recognized as sufficiently describing and enabling the same range being
broken down into at least equal halves, thirds, quarters, fifths, tenths,
etc. As a non-limiting example, each range discussed herein can be
readily broken down into a lower third, middle third and upper third,
etc. As will also be understood by one skilled in the art all language
such as "up to," "at least," "greater than," "less than," and the like,
include the number recited and refer to ranges which can be subsequently
broken down into subranges as discussed above. Finally, as will be
understood by one skilled in the art, a range includes each individual
member. Thus, for example, a group having 1-3 peptides refers to groups
having 1, 2, or 3 peptides Similarly, a group having 1-5 peptides refers
to groups having 1, 2, 3, 4, or 5 peptides, and so forth.

[0282] All patents, patent applications, provisional applications, and
publications referred to or cited herein are incorporated by reference in
their entirety, including all figures and tables, to the extent they are
not inconsistent with the explicit teachings of this specification.